The invention relates to a method of analyzing a polynucleotide sequence. This invention is also directed to diagnostic aids for analyzing the nucleic acid composition and content of biological samples, including samples derived from medical and agricultural sources.
The genomics revolution has spurned a significant interest in the creation of personalized drugs based on the genetic constitution, or xe2x80x98genotypexe2x80x99 of the recipient and in related scientific research areas such as high throughput drug discovery, diagnostics in human subjects, animal health and cloning, and the genetic selection of plants with desired traits. Consequently, robust technology platforms have been created for the rapid identification of genomics sequence and the creation of sequence databases wherefrom making electronic comparisons can identify even small differences in sequences between individuals (such as, single nucleotide polymorphisms, or SNPs). It is estimated that the xcx9c80,000 human genes contain about 200,000 cSNPs, (i.e., SNPs that lie in coding regions) whereas over several million randomly chosen SNPs are likely to be detected in the genome.
It has been suggested that testing a dense panel of SNPs arrayed across both coding and noncoding regions, termed xe2x80x98ancestral haplotypesxe2x80x99, in affected individuals and in controls may be an appropriate strategy to identify associations that narrowly locate the neighborhood of a susceptibility or resistance gene. A dense map of SNPs with at least 100,000 SNPs is estimated to satisfy the resolution required.
Recent novel approaches to assessing DNA sequence differences between individuals, such as, minisequencing, multiplex reverse dot blots, DNA microarrays, and the TaqMan approach have lent a significant impetus to the speed, cost efficiency and accuracy of association studies.
Other strategies described in the art for identification of polymorphisms have included a genome simplification or complexity reduction step, including construction of shotgun libraries in BACs, PACs, or cosmids using DNA samples from a limited number of subjects. Clones from the library are sequenced. Alternatively, hybridization or enzymatic approaches (such as RDA or mismatch scanning using bacterial proteins or enzymes involved in homologous recombination) have been used. These latter approaches are rapid and cost effective and eliminate the need for sequencing extensive regions of genomic DNA for identifying polymorphisms.
Yet, the above methods suffer from 2 major disadvantages, a) lack of throughput, and b) inadequate representation of the genome per iteration. As such, there is a need for further improvements in the technology for high throughput genomics analytical methods, in particular, detection of SNPs.
This invention features methods for sensitively detecting the presence of a nucleic acid in a sample. Described herein are methods for synergistic multiplexed amplifications of nucleic acids. Multiple individual chemical and biochemical reactions for target identification, amplification and partitioning and detection of each signal independently of other similar signals in the multiplexed reaction can be caused to occur simultaneously.
Consequently, it is an object of this invention to provide a method for identifying nucleic acids in a sample.
It is an object of this invention to quantify nucleic acids from a sample.
It is an object of this invention to rapidly and cost-effectively determine the quantitative presence of nucleic acids in a sample.
It is an object of this invention to provide methods for the multiplexed analysis of nucleic acids from a sample.
It is an object of this invention to identify qualitative differences between nucleic acids of two or more organisms of the same species.
It is an object of this invention to provide a method for comparing the amount of nucleic acids present in two or more samples.
It is an object of this invention to identify an organism by identifying presence of the genomic nucleic acids of the organism in a sample.
It is an object of this invention to quantify the presence of nucleic acids of an organism relative to the amount of nucleic acids of another organism present in the same sample.
It is an object of this invention to identify the physiological state of an organism by quantifying gene expression in the sample.
In one aspect, the invention features a method of analyzing a polynucleotide, e.g., detecting a genetic event, e.g., a single nucleotide polymorphism, in a sample.
The method includes:
1. providing a sample which includes a sample polynucleotide sequence to be analyzed, said sample polynucleotide being at least partially single stranded and containing a region of recognizable sequence adjacent to the single stranded terminus;
2. (a) annealing an effective amount of sample sequence to a hairpin primer, wherein the hairpin primer comprises at least one copy of a nucleotide sequence complementary to the sequence of the sample polynucleotide sequence and optionally,
(b) extending the hairpin primer with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme to yield a product, e.g., a single stranded polymerase extended product complementary to a substantial portion of the single stranded region of the sample polynucleotide; and
(c) optionally, amplifying the polymerase extended product to produce amplified products, herein termed amplified products;
3. analyzing said product from 2 (a, b or c) by providing an array of a plurality of capture probes, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and wherein each positionally distinguishable capture probe includes at least one unique (i.e., not repeated in another capture probe) region, optionally, adjacent to a common (repeated in all the capture probes) region;
hybridizing the amplified products with the array of capture probes, thereby analyzing the sample sequence.
In preferred embodiments, analyzing a sample polynucleotide includes, e.g., sequencing the polynucleotide sequence, e.g., by sequencing by hybridization or positional sequencing by hybridization, detecting the presence of, or identifying a genetic event, e.g., a SNP, in a target nucleic acid, e.g., a DNA.
In preferred embodiments, the genetic event is within 12 bases, more preferably within 1, 2, 3, 4, 5, or 6 bases in the single stranded portion of sample polynucleotide that is adjacent to and abuts the double stranded region; more specifically, in the region of the single stranded portion of the sample polynucleotide farthest from the single stranded terminus, or is sufficiently close to the double strand portion of the sample polynucleotide that a mismatch would inhibit DNA polymerase-based extension from the sample polynucleotide/hairpin primer complex.
In preferred embodiments, the genetic event is located anywhere within the single stranded portion of the sample polynucleotide.
In preferred embodiments, the target polynucleotide in the sample is amplified, e.g., by PCR, prior to contact with a hairpin primer.
In preferred embodiments, the hairpin primer includes a site for a type 2s restriction enzyme and the site is positioned, e.g., such that a type 2s restriction enzyme binding at the site cleaves adjacent the region which binds the sample sequence or cleaves in the region which binds the sample sequence.
In a preferred embodiment, a region of the hairpin primer is complementary to a genetic event, e.g., a mutation or SNP, and hybridizes effectually to sample nucleic acid having the event and sample nucleic acid not having the event.
In preferred embodiments the polynucleotide sequence is: a DNA molecule: all or part of a known gene; wild type DNA; mutant DNA; a genomic fragment, particularly a human genomic fragment; a cDNA, particularly a human cDNA.
In preferred embodiments the polynucleotide sequence is: an RNA molecule: nucleic acids derived from RNA transcripts; wild type RNA; mutant RNA, particularly a human RNA.
In preferred embodiments the polynucleotide sequence is: a human sequence; a non-human sequence, e.g., a mouse, rat, pig, primate.
In preferred embodiments the method is performed: on a sample from a human subject; and a sample from a prenatal subject, as part of genetic counseling; to determine if the individual from whom the target nucleic acid is taken should receive a drug or other treatment; to diagnose an individual for a disorder or for predisposition to a disorder; to stage a disease or disorder.
In preferred embodiments the capture probes are single stranded probes in an array.
In preferred embodiments the capture probes have a structure comprising a double stranded portion and a single stranded portion in an array.
In preferred embodiments hybridization to the array is detected by mass spectrophotometry, e.g., by MALDI-TOF mass spectrophotometry.
In a preferred embodiment the amplified product, which hybridizes to a capture probe, or the capture probe, is the substrate of or template for an enzyme mediated reaction.
For example, after hybridization to the capture probe, the amplified product is ligated to the capture probe, or after the hybridization it is extended along the capture probe.
In preferred embodiments the method includes one or more enzyme mediated reactions in which a nucleic acid used in the method, e.g., an amplified product, a capture probe, a sequence to be analyzed, or a molecule which hybridizes thereto, is a substrate or template for the enzyme mediated reaction. The enzyme mediated reaction can be: an extension reaction, e.g., a reaction catalyzed by a polymerase; a linking reaction, e.g., a ligation, e.g., a reaction catalyzed by a ligase; or a nucleic acid cleavage reaction, e.g., a cleavage catalyzed by a restriction enzyme, e.g., a Type IIs enzyme or an exonuclease. The amplified product, which hybridizes with the capture probe, can be the substrate in an enzyme mediated reaction, e.g., it can be ligated to a strand of the capture probe. Alternatively, the capture probe can be extended along the hybridized amplified products. (Any of the extension reactors discussed herein can be performed with labeled, or chain terminating, subunits). The capture probe duplex can be the substrate for a cleavage reaction. These reactions can be used to increase the specificity of the method or to otherwise aid in detection, e.g., by providing a signal.
Methods of U.S. Pat. No. 5,503,980 and U.S. Pat. No. 5,631,134, both of which are hereby incorporated by reference can be used herein, particularly, the array and array-related steps recited herein can use methods taught in these patents.
In preferred embodiments, the method includes providing an array having a plurality of capture probes, wherein each of the capture probes is a) positionally distinguishable from the other capture probes of the plurality and has a unique variable region (not repeated in another capture probe of the plurality), b) has a variable region capable of hybridizing adjacent to the genetic event; and has a 3xe2x80x2 end capable of serving as a priming site for extension; hybridizing the amplified products having a genetic event to a capture probe of the array, (preferably the region of the amplified products having a genetic event hybridizes adjacent to the variable region of a capture probe); and using the 3xe2x80x2 end of the capture probe to extend across the region of genomic nucleic acid having a genetic event with one or more terminating base species, where if more than one is used each has a unique distinguishable label, e.g., label 1 for base A, label 2 for base T, label 3 for base G, and label 4 for base C; thereby analyzing the amplified products.
In a preferred embodiment, at least one reaction step is performed on a three- dimensional array, e.g., a gel array.
Preferably, a hairpin primer has about 15-1500 nucleotides, and more preferably about 24-500 nucleotides and most preferably about 30-150 nucleotides.
The hairpin primer is constructed of DNA or RNA or analogs thereof. Preferably, the hairpin primer is constructed of DNA.
The polymerase enzyme can be any that affects the synthesis of the polymerase extended product.
In another aspect, the invention features a method of analyzing a polynucleotide, e.g., detecting a genetic event, e.g., a single nucleotide polymorphism, in a sample.
The method includes:
1. providing a sample which includes a sample polynucleotide sequence to be analyzed, said sample polynucleotide being at least partially single stranded and containing a region of recognizable sequence adjacent to the single stranded terminus;
2. (a) annealing an effective amount of sample sequence to a hairpin primer, wherein the hairpin primer comprises at least one copy of a nucleotide sequence complementary to the sequence of the sample polynucleotide sequence and optionally,
(b) extending the hairpin primer with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme to yield a product, e.g., a single stranded polymerase extended product complementary to a substantial portion of the single stranded region of the sample polynucleotide; and
(c) optionally, amplifying the polymerase extended product to produce amplified products, herein termed amplified products;
3. analyzing said product from 2 (b) or (c) by providing a separation means, e.g., a gel for electrophoretic separation of nucleic acid fragments, e.g., polyacrylamide gel, agarose gel, capillary electrophoresis or other described in the art.
In preferred embodiments, analyzing a sample polynucleotide includes, e.g., sequencing the polynucleotide sequence, e.g., by sequencing by hybridization or positional sequencing by hybridization, detecting the presence of, or identifying a genetic event, e.g., a SNP, in a target nucleic acid, e.g., a DNA.
In preferred embodiments, the genetic event is within 12 bases, more preferably within 1, 2, 3, 4, 5, or 6 bases in the single stranded portion of sample polynucleotide that is adjacent to and immediately follows the double stranded region; more specifically, in the region of the single stranded portion of the sample polynucleotide farthest from the single stranded terminus, or is sufficiently close to the double strand portion of the sample polynucleotide that a mismatch would inhibit DNA polymerase-based extension from the sample polynucleotide/hairpin primer complex.
In equally preferred embodiments, the genetic event is located anywhere within the single stranded portion of the sample polynucleotide.
In preferred embodiments, the target polynucleotide in the sample is amplified, e.g., by PCR, prior to contact with a hairpin primer.
In preferred embodiments, the hairpin primer includes a site for a type 2s restriction enzyme and the site is positioned, e.g., such that a type 2s restriction enzyme binding at the site cleaves adjacent the region which binds the sample sequence or cleaves in the region which binds the sample sequence.
In a preferred embodiment, a region of the hairpin primer is complementary to a genetic event, e.g., a mutation or SNP, and hybridizes effectually to sample nucleic acid having the event and sample nucleic acid not having the event.
In preferred embodiments the polynucleotide sequence is: a DNA molecule: all or part of a known gene; wild type DNA; mutant DNA; a genomic fragment, particularly a human genomic fragment; a cDNA, particularly a human cDNA.
In preferred embodiments the polynucleotide sequence is: an RNA molecule: nucleic acids derived from RNA transcripts; wild type RNA; mutant RNA, particularly a human RNA.
In preferred embodiments the polynucleotide sequence is: a human sequence; a non-human sequence, e.g., a mouse, rat, pig, primate.
In preferred embodiments the method is performed: on a sample from a human subject; and a sample from a prenatal subject, as part of genetic counseling; to determine if the individual from whom the target nucleic acid is taken should receive a drug or other treatment; to diagnose an individual for a disorder or for predisposition to a disorder; to stage a disease or disorder.
In preferred embodiments the capture probes are single stranded probes in an array.
In preferred embodiments the capture probes have a structure comprising a double stranded portion and a single stranded portion in an array.
In preferred embodiments hybridization to the array is detected by mass spectrophotometry, e.g., by MALDI-TOF mass spectrophotometry.
In a preferred embodiment the amplified products (amplified products) which hybridizes to a capture probe, or the capture probe, is the substrate of or template for an enzyme mediated reaction.
For example, after hybridization to the capture probe, the amplified products is ligated to the capture probe, or after the hybridization it is extended along the capture probe.
In preferred embodiments the method includes one or more enzyme mediated reactions in which a nucleic acid used in the method, e.g., an amplified products, a capture probe, a sequence to be analyzed, or a molecule which hybridizes thereto, is a substrate or template for the enzyme mediated reaction. The enzyme mediated reaction can be: an extension reaction, e.g., a reaction catalyzed by a polymerase; a linking reaction, e.g., a ligation, e.g., a reaction catalyzed by a ligase; or a nucleic acid cleavage reaction, e.g., a cleavage catalyzed by a restriction enzyme, e.g., a Type IIs enzyme or an exonuclease. The amplified products which hybridizes with the capture probe can be the substrate in an enzyme mediated reaction, e.g., it can be ligated to a strand of the capture probe. Alternatively, the capture probe can be extended along the hybridized amplified products. (Any of the extension reactors discussed herein can be performed with labeled, or chain terminating, subunits). The capture probe duplex can be the substrate for a cleavage reaction. These reactions can be used to increase the specificity of the method or to otherwise aid in detection, e.g., by providing a signal.
Methods of U.S. Pat. No. 5,503,980 and U.S. Pat. No. 5,631,134, both of which are hereby incorporated by reference can be used herein, particularly, the array and array-related steps recited herein can use methods taught in these patents.
In preferred embodiments, the method includes providing an array having a plurality of capture probes, wherein each of the capture probes is a) positionally distinguishable from the other capture probes of the plurality and has a unique variable region (not repeated in another capture probe of the plurality), b) has a variable region capable of hybridizing adjacent to the genetic event; and has a 3xe2x80x2 end capable of serving as a priming site for extension; hybridizing the amplified products having a genetic event to a capture probe of the array, (preferably the region of the amplified products having a genetic event hybridizes adjacent to the variable region of a capture probe); and using the 3xe2x80x2 end of the capture probe to extend across the region of genomic nucleic acid having a genetic event with one or more terminating base species, where if more than one is used each has a unique distinguishable label, e.g., label 1 for base A, label 2 for base T, label 3 for base G, and label 4 for base C; thereby analyzing the amplified products.
In a preferred embodiment, at least one reaction step is performed on a three-dimensional array, e.g., a gel array.
Preferably, a hairpin primer has about 15-1500 nucleotides, and more preferably about 24-500 nucleotides and most preferably about 30-150 nucleotides.
The hairpin primer is constructed of DNA or RNA or analogs thereof. Preferably, the hairpin primer is constructed of DNA.
The polymerase enzyme can be any that affects the synthesis of the polymerase extended product.
In another aspect, the invention features a method of analyzing a polynucleotide, e.g., detecting a genetic event, e.g., a single nucleotide polymorphism, in a sample.
The method includes:
1. providing a sample which includes a sample polynucleotide sequence to be analyzed, said sample polynucleotide being at least partially single stranded and containing a region of recognizable sequence adjacent to the single stranded terminus;
2. (a) annealing an effective amount of sample sequence to a hairpin primer, wherein the hairpin primer comprises at least one copy of a nucleotide sequence complementary to the sequence of the sample polynucleotide sequence and optionally,
(b) extending the hairpin primer with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme to yield a product, e.g., a single stranded polymerase extended product complementary to a substantial portion of the single stranded region of the sample polynucleotide; and
(c) optionally, amplifying the polymerase extended product to produce amplified products, herein termed amplified products;
(d) cleaving the amplified products with a restriction enzyme, e.g., a type IIs restriction enzyme;
3. analyzing said cleaved product from 3 by providing an array of a plurality of capture probes, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and wherein each positionally distinguishable capture probe includes at least one unique (i.e., not repeated in another capture probe) region, optionally, adjacent to a common (repeated in all the capture probes) region;
hybridizing the amplified products with the array of capture probes, thereby analyzing the sample sequence.
In preferred embodiments, analyzing a sample polynucleotide includes, e.g., sequencing the polynucleotide sequence, e.g., by sequencing by hybridization or positional sequencing by hybridization, detecting the presence of, or identifying a genetic event, e.g., a SNP, in a target nucleic acid, e.g., a DNA.
In preferred embodiments, the genetic event is within 12 bases, more preferably within 1, 2, 3, 4, 5, or 6 bases in the single stranded portion of sample polynucleotide that is adjacent to and immediately follows the double stranded region; more specifically, in the region of the single stranded portion of the sample polynucleotide farthest from the single stranded terminus, or is sufficiently close to the double strand portion of the sample polynucleotide that a mismatch would inhibit DNA polymerase-based extension from the sample polynucleotide/hairpin primer complex.
In equally preferred embodiments, the genetic event is located anywhere within the single stranded portion of the sample polynucleotide.
In preferred embodiments, the target polynucleotide in the sample is amplified, e.g., by PCR, prior to contact with a hairpin primer.
In preferred embodiments, the hairpin primer includes a site for a type 2s restriction enzyme and the site is positioned, e.g., such that a type 2s restriction enzyme binding at the site cleaves adjacent the region which binds the sample sequence or cleaves in the region which binds the sample sequence.
In a preferred embodiment, a region of the hairpin primer is complementary to a genetic event, e.g., a mutation or SNP, and hybridizes effectually to sample nucleic acid having the event and sample nucleic acid not having the event.
In preferred embodiments the polynucleotide sequence is: a DNA molecule: all or part of a known gene; wild type DNA; mutant DNA; a genomic fragment, particularly a human genomic fragment; a cDNA, particularly a human cDNA.
In preferred embodiments the polynucleotide sequence is: an RNA molecule: nucleic acids derived from RNA transcripts; wild type RNA; mutant RNA, particularly a human RNA.
In preferred embodiments the polynucleotide sequence is: a human sequence; a non-human sequence, e.g., a mouse, rat, pig, primate.
In preferred embodiments the method is performed: on a sample from a human subject; and a sample from a prenatal subject, as part of genetic counseling; to determine if the individual from whom the target nucleic acid is taken should receive a drug or other treatment; to diagnose an individual for a disorder or for predisposition to a disorder; to stage a disease or disorder.
In preferred embodiments the capture probes are single stranded probes in an array.
In preferred embodiments the capture probes have a structure comprising a double stranded portion and a single stranded portion in an array.
In preferred embodiments hybridization to the array is detected by mass spectrophotometry, e.g., by MALDI-TOF mass spectrophotometry.
In a preferred embodiment the amplified sample that hybridizes to a capture probe, or the capture probe, is the substrate of or template for an enzyme mediated reaction.
For example, after hybridization to the capture probe, the amplified products is ligated to the capture probe, or after the hybridization it is extended along the capture probe.
In preferred embodiments the method includes one or more enzyme mediated reactions in which a nucleic acid used in the method, e.g., an amplified products, a capture probe, a sequence to be analyzed, or a molecule which hybridizes thereto, is a substrate or template for the enzyme mediated reaction. The enzyme mediated reaction can be: an extension reaction, e.g., a reaction catalyzed by a polymerase; a linking reaction, e.g., a ligation, e.g., a reaction catalyzed by a ligase; or a nucleic acid cleavage reaction, e.g., a cleavage catalyzed by a restriction enzyme, e.g., a Type IIs enzyme or an exonuclease. The amplified products that hybridizes with the capture probe can be the substrate in an enzyme-mediated reaction, e.g., it can be ligated to a strand of the capture probe. Alternatively, the capture probe can be extended along the hybridized amplified products. (Any of the extension reactors discussed herein can be performed with labeled, or chain terminating, subunits). The capture probe duplex can be the substrate for a cleavage reaction. These reactions can be used to increase the specificity of the method or to otherwise aid in detection, e.g., by providing a signal.
Methods of U.S. Pat. No. 5,503,980 and U.S. Pat. No. 5,631,134, both of which are hereby incorporated by reference can be used herein, particularly, the array and array-related steps recited herein can use methods taught in these patents.
In preferred embodiments, the method includes providing an array having a plurality of capture probes, wherein each of the capture probes is a) positionally distinguishable from the other capture probes of the plurality and has a unique variable region (not repeated in another capture probe of the plurality), b) has a variable region capable of hybridizing adjacent to the genetic event; and has a 3xe2x80x2 end capable of serving as a priming site for extension; hybridizing the amplified products having a genetic event to a capture probe of the array, (preferably the region of the amplified products having a genetic event hybridizes adjacent to the variable region of a capture probe); and using the 3xe2x80x2 end of the capture probe to extend across the region of genomic nucleic acid having a genetic event with one or more terminating base species, where if more than one is used each has a unique distinguishable label, e.g., label 1 for base A, label 2 for base T, label 3 for base G, and label 4 for base C; thereby analyzing the amplified products.
In a preferred embodiment, at least one reaction step is performed on a three-dimensional array, e.g., a gel array.
Preferably, a hairpin primer has about 15-1500 nucleotides, and more preferably about 24-500 nucleotides and most preferably about 30-150 nucteotides.
The hairpin primer is constructed of DNA or RNA or analogs thereof. Preferably, the hairpin primer is constructed of DNA.
The polymerase enzyme can be any that affects the synthesis of the polymerase extended product.
In another aspect, the invention features a method of analyzing a polynucleotide, e.g., detecting a genetic event, e.g., a single nucleotide polymorphism, in a sample.
The method includes:
1. providing a sample which includes a sample polynucleotide sequence to be analyzed, said sample polynucleotide being at least partially single stranded and containing a region of recognizable sequence adjacent to the single stranded terminus;
2. (a) annealing an effective amount of sample sequence to a hairpin primer, wherein the hairpin primer comprises at least one copy of a nucleotide sequence complementary to the sequence of the sample polynucleotide sequence and optionally,
(b) extending the hairpin primer with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme to yield a product, e.g., a single stranded polymerase extended product complementary to a substantial portion of the single stranded region of the sample polynucleotide; and
(c) optionally, amplifying the polymerase extended product to produce amplified products, herein termed amplified products;
(e) cleaving the amplified products with a restriction enzyme, e.g., a type IIs restriction enzyme;
3. (a) annealing an effective amount of cleaved amplified products (from 2e) to at least one copy of a positioning oligonucleotide having a 5xe2x80x2 nucleotide sequence complementary to a portion of the sequence comprising the 3xe2x80x2 end of the cleaved amplified products and a 3xe2x80x2 nucleotide sequence complementary to a portion of the sequence comprising the 5xe2x80x2 end of the cleaved amplified products wherein the 5xe2x80x2 end and the 3xe2x80x2 end of the cleaved amplified products are positioned so as to abut each other; and
(b) joining the 5xe2x80x2 end and the 3xe2x80x2 end of the cleaved amplified products to yield a circular oligonucleotide template and optionally,
(c) combining the circular template with an effective amount of a RCA primer, at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme to yield a product, e.g., a single stranded oligonucleotide multimer complementary to the circular oligonucleotide template; and
(d) optionally, cleaving the product to produce cleaved amplified product
4. analyzing product from 3 b, c, or d, by providing an array of a plurality of capture probes, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and wherein each positionally distinguishable capture probe includes at least one unique (i.e., not repeated in another capture probe) region, optionally, adjacent to a common (i.e., repeated in all the capture probes) region;
hybridizing the amplified products with the array of capture probes, thereby analyzing the sample sequence.
In preferred embodiments, analyzing a sample polynucleotide includes, e.g., sequencing the polynucleotide sequence, e.g., by sequencing by hybridization or positional sequencing by hybridization, detecting the presence of, or identifying a genetic event, e.g., a SNP, in a target nucleic acid, e.g., a DNA.
In preferred embodiments, the genetic event is within 12 bases, more preferably within 1, 2, 3, 4, 5, or 6 bases in the single stranded portion of sample polynucleotide that is adjacent to and immediately follows the double stranded region; more specifically, in the region of the single stranded portion of the sample polynucleotide farthest from the single stranded terminus, or is sufficiently close to the double strand portion of the sample polynucleotide that a mismatch would inhibit DNA polymerase-based extension from the sample polynucleotide/hairpin primer complex.
In equally preferred embodiments, the genetic event is located anywhere within the single stranded portion of the sample polynucleotide.
In preferred embodiments, the target polynucleotide in the sample is amplified, e.g., by PCR, prior to contact with a hairpin primer.
In preferred embodiments, the hairpin primer includes a site for a type 2s restriction enzyme and the site is positioned, e.g., such that a type 2s restriction enzyme binding at the site cleaves adjacent the region which binds the sample sequence or cleaves in the region which binds the sample sequence.
In a preferred embodiment, a region of the hairpin primer is complementary to a genetic event, e.g., a mutation or SNP, and hybridizes effectually to sample nucleic acid having the event and sample nucleic acid not having the event.
In preferred embodiments the polynucleotide sequence is: a DNA molecule: all or part of a known gene; wild type DNA; mutant DNA; a genomic fragment, particularly a human genomic fragment; a cDNA, particularly a human cDNA.
In preferred embodiments the polynucleotide sequence is: an RNA molecule: nucleic acids derived from RNA transcripts; wild type RNA; mutant RNA, particularly a human RNA.
In preferred embodiments the polynucleotide sequence is: a human sequence; a non-human sequence, e.g., a mouse, rat, pig, primate.
In preferred embodiments the method is performed: on a sample from a human subject; and a sample from a prenatal subject, as part of genetic counseling; to determine if the individual from whom the target nucleic acid is taken should receive a drug or other treatment; to diagnose an individual for a disorder or for predisposition to a disorder; to stage a disease or disorder.
In preferred embodiments the capture probes are single stranded probes in an array.
In preferred embodiments the capture probes have a structure comprising a double stranded portion and a single stranded portion in an array.
In preferred embodiments hybridization to the array is detected by mass spectrophotometry, e.g., by MALDI-TOF mass spectrophotometry.
In a preferred embodiment the amplified products (amplified products) which hybridizes to a capture probe, or the capture probe, is the substrate of or template for an enzyme mediated reaction.
For example, after hybridization to the capture probe, the amplified products is ligated to the capture probe, or after the hybridization it is extended along the capture probe.
In preferred embodiments the method includes one or more enzyme mediated reactions in which a nucleic acid used in the method, e.g., an amplified products, a capture probe, a sequence to be analyzed, or a molecule which hybridizes thereto, is a substrate or template for the enzyme mediated reaction. The enzyme mediated reaction can be: an extension reaction, e.g., a reaction catalyzed by a polymerase; a linking reaction, e.g., a ligation, e.g., a reaction catalyzed by a ligase; or a nucleic acid cleavage reaction, e.g., a cleavage catalyzed by a restriction enzyme, e.g., a Type IIs enzyme or an exonuclease. The amplified products which hybridizes with the capture probe can be the substrate in an enzyme mediated reaction, e.g., it can be ligated to a strand of the capture probe. Alternatively, the capture probe can be extended along the hybridized amplified products. (Any of the extension reactors discussed herein can be performed with labeled, or chain terminating, subunits). The capture probe duplex can be the substrate for a cleavage reaction. These reactions can be used to increase the specificity of the method or to otherwise aid in detection, e.g., by providing a signal.
Methods of U.S. Pat. No. 5,503,980 and U.S. Pat. No. 5,631,134, both of which are hereby incorporated by reference can be used herein, particularly, the array and array-related steps recited herein can use methods taught in these patents.
In preferred embodiments, the method includes providing an array having a plurality of capture probes, wherein each of the capture probes is a) positionally distinguishable from the other capture probes of the plurality and has a unique variable region (not repeated in another capture probe of the plurality), b) has a variable region capable of hybridizing adjacent to the genetic event; and has a 3xe2x80x2 end capable of serving as a priming site for extension; hybridizing the amplified products having a genetic event to a capture probe of the array, (preferably the region of the amplified products having a genetic event hybridizes adjacent to the variable region of a capture probe); and using the 3xe2x80x2 end of the capture probe to extend across the region of genomic nucleic acid having a genetic event with one or more terminating base species, where if more than one is used each has a unique distinguishable label, e.g., label 1 for base A, label 2 for base T, label 3 for base G, and label 4 for base C; thereby analyzing the amplified products.
In a preferred embodiment, at least one reaction step is performed on a three-dimensional array, e.g., a gel array.
Preferably, a hairpin primer has about 15-1500 nucleotides, and more preferably about 24-500 nucleotides and most preferably about 30-150 nucleotides.
The hairpin primer is constructed of DNA or RNA or analogs thereof. Preferably, the hairpin primer is constructed of DNA.
The polymerase enzyme can be any that affects the synthesis of the polymerase extended product.
In another aspect, the invention features a method of analyzing a polynucleotide, e.g., detecting a genetic event, e.g., a single nucleotide polymorphism, in a sample.
The method includes:
1. providing a sample which includes a sample polynucleotide sequence to be analyzed, said sample polynucleotide being at least partially single stranded and containing a region of recognizable sequence adjacent to the single stranded terminus;
2. (a) annealing an effective amount of sample sequence to a hairpin primer, wherein the hairpin primer comprises at least one copy of a nucleotide sequence complementary to the sequence of the sample polynucleotide sequence and optionally,
(b) extending the hairpin primer with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme to yield a product, e.g., a single stranded polymerase extended product complementary to a substantial portion of the single stranded region of the sample polynucleotide; and
(c) optionally, amplifying the polymerase extended product to produce amplified products, herein termed amplified products;
(d) hybridizing a first set of amplified products derived from a first sample polynucleotide(s) with a second set of amplified products derived from a second sample polynucleotide(s);
(e) separating duplexes from (d) containing mismatches from perfectly matched duplexes;
3. analyzing mismatched duplexes from (e) by providing an array of a plurality of capture probes, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and wherein each positionally distinguishable capture probe includes at least one unique (i.e., not repeated in another capture probe) region, optionally, adjacent to a common (repeated in all the capture probes) region; thereby analyzing the sample sequence.
In preferred embodiments, analyzing a sample polynucleotide includes, e.g., sequencing the polynucleotide sequence, e.g., by sequencing by hybridization or positional sequencing by hybridization, detecting the presence of, or identifying a genetic event, e.g., a SNP, in a target nucleic acid, e.g., a DNA.
In preferred embodiments, the genetic event is within 12 bases, more preferably within 1, 2, 3, 4, 5, or 6 bases in the single stranded portion of sample polynucleotide that is adjacent to and immediately follows the double stranded region; more specifically, in the region of the single stranded portion of the sample polynucleotide farthest from the single stranded terminus, or is sufficiently close to the double strand portion of the sample polynucleotide that a mismatch would inhibit DNA polymerase-based extension from the sample polynucleotide/hairpin primer complex.
In preferred embodiments, the genetic event is located anywhere within the single stranded portion of the sample polynucleotide.
In preferred embodiments, the target polynucleotide in the sample is amplified, e.g., by PCR, prior to contact with a hairpin primer.
In preferred embodiments, the hairpin primer includes a site for a type 2s restriction enzyme and the site is positioned, e.g., such that a type 2s restriction enzyme binding at the site cleaves adjacent the region which binds the sample sequence or cleaves in the region which binds the sample sequence.
In a preferred embodiment, a region of the hairpin primer is complementary to a genetic event, e.g., a mutation or SNP, and hybridizes effectually to sample nucleic acid having the event and sample nucleic acid not having the event.
In preferred embodiments the polynucleotide sequence is: a DNA molecule: all or part of a known gene; wild type DNA; mutant DNA; a genomic fragment, particularly a human genomic fragment; a cDNA, particularly a human cDNA.
In preferred embodiments the polynucleotide sequence is: an RNA molecule: nucleic acids derived from RNA transcripts; wild type RNA; mutant RNA, particularly a human RNA.
In preferred embodiments the polynucleotide sequence is: a human sequence; a non-human sequence, e.g., a mouse, rat, pig, primate.
In preferred embodiments the method is performed: on a sample from a human subject; and a sample from a prenatal subject, as part of genetic counseling; to determine if the individual from whom the target nucleic acid is taken should receive a drug or other treatment; to diagnose an individual for a disorder or for predisposition to a disorder; to stage a disease or disorder.
In preferred embodiments the capture probes are single stranded probes in an array.
In preferred embodiments the capture probes have a structure comprising a double stranded portion and a single stranded portion in an array.
In preferred embodiments hybridization to the array is detected by mass spectrophotometry, e.g., by MALDI-TOF mass spectrophotometry.
In a preferred embodiment the amplified products (amplified products) which hybridizes to a capture probe, or the capture probe, is the substrate of or template for an enzyme mediated reaction.
For example, after hybridization to the capture probe, the amplified products is ligated to the capture probe, or after the hybridization it is extended along the capture probe.
In preferred embodiments the method includes one or more enzyme mediated reactions in which a nucleic acid used in the method, e.g., an amplified products, a capture probe, a sequence to be analyzed, or a molecule which hybridizes thereto, is a substrate or template for the enzyme mediated reaction. The enzyme mediated reaction can be: an extension reaction, e.g., a reaction catalyzed by a polymerase; a linking reaction, e.g., a ligation, e.g., a reaction catalyzed by a ligase; or a nucleic acid cleavage reaction, e.g., a cleavage catalyzed by a restriction enzyme, e.g., a Type IIs enzyme or an exonuclease. The amplified products which hybridizes with the capture probe can be the substrate in an enzyme mediated reaction, e.g., it can be ligated to a strand of the capture probe. Alternatively, the capture probe can be extended along the hybridized amplified products. (Any of the extension reactors discussed herein can be performed with labeled, or chain terminating, subunits). The capture probe duplex can be the substrate for a cleavage reaction. These reactions can be used to increase the specificity of the method or to otherwise aid in detection, e.g., by providing a signal.
Methods of U.S. Pat. No. 5,503,980 and U.S. Pat. No. 5,631,134, both of which are hereby incorporated by reference can be used herein, particularly, the array and array-related steps recited herein can use methods taught in these patents.
In preferred embodiments, the method includes providing an array having a plurality of capture probes, wherein each of the capture probes is a) positionally distinguishable from the other capture probes of the plurality and has a unique variable region (not repeated in another capture probe of the plurality), b) has a variable region capable of hybridizing adjacent to the genetic event; and has a 3xe2x80x2 end capable of serving as a priming site for extension; hybridizing the amplified products having a genetic event to a capture probe of the array, (preferably the region of the amplified products having a genetic event hybridizes adjacent to the variable region of a capture probe); and using the 3xe2x80x2 end of the capture probe to extend across the region of genomic nucleic acid having a genetic event with one or more terminating base species, where if more than one is used each has a unique distinguishable label, e.g., label 1 for base A, label 2 for base T, label 3 for base G, and label 4 for base C; thereby analyzing the amplified products.
In a preferred embodiment, at least one reaction step is performed on a three-dimensional array, e.g., a gel array.
Preferably, a hairpin primer has about 15-1500 nucleotides, and more preferably about 24-500 nucleotides and most preferably about 30-150 nucleotides.
The hairpin primer is constructed of DNA or RNA or analogs thereof. Preferably, the hairpin primer is constructed of DNA.
The polymerase enzyme can be any that affects the synthesis of the polymerase extended product.
In another aspect, the invention features, a hairpin primer. The probe includes, preferably in a 5xe2x80x2 to 3xe2x80x2 orientation:
a first nucleic acid contacting region which is complementary to a first portion of a target nucleic acid;
a double stranded region, comprised of complementary base pairing of a portion of the hairpin primer oligonucleotide near its 5xe2x80x2 end with a portion of the same oligonucleotide near its 3xe2x80x2 end;
optionally, at least one single-stranded region;
a second nucleic acid contacting region that is complementary to a second portion of the target nucleic acid.
In preferred embodiments, the elements are in the recited order.
In specific embodiments, one or more nucleotides in the first nucleotide contacting region or second nucleotide contacting region may be designed to contain degenerate nucleotides.
In preferred embodiments, the first nucleotide contacting region is 4-12 nucleotides long.
In preferred embodiments, the double stranded region is 4-20 nucleotides long.
In preferred embodiments, the single stranded region is 4-12 bases long.
In preferred embodiments, the second nucleic acid contacting region is 4-12 bases long.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable materials and methods are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.